The invention concerns a device for the manipulation and treatment of biological objects by means of electrical pulses, particularly for the permeation (poration) and/or fusion of cells or of synthetic formations enclosed in a membrane, such as liposomes or vesicles, or for the permeation of membrane or film materials in miniaturized electrode structures, and manipulation or treatment processes using a device of this type.
For specific biotechnology, medical, or genetic technology tasks, the temporary and reversible increase of the permeability of the envelope of living cells suspended in the fluid is of interest (overview in xe2x80x9cElectromanipulation of Cellsxe2x80x9d, U. Zimmerman, G. A. Neil, CRC, 1996). Besides chemical, virus-based, and laser-induced permeation methods, due to the simplicity and precision of the application, permeation by means of short electrical pulses (electroporation or electropermeation) has developed, with pulsed direct current signals (cf. U. Zimmerman et al. in xe2x80x9cBBAxe2x80x9d, vol. 641, 1982, p. 160 et seq) or choppered HF fields (cf. PCT/US88/03457) being used. In the generally known electroporation devices commercially available until now, the electroporations and/or fusions are performed in chambers with electrodes whose dimensions are significantly larger than the dimensions of the objects treated, with the following disadvantages thereby arising.
Until now, cells could not be permeated in culture media, because these have a high conductivity and, due to the low dielectric constants and conductivity of biological cells, the electrical fields would be formed outside the cells. In addition, the culture media would lead to a high thermal stress of the cells to be treated through resistance heating because of the current flow through the culture medium. The field of application of the typical electroporation devices is furthermore restricted to robust and resistant cells. In addition, optimization of the fusion parameters such as the field strength and pulse frequency is only possible in a restricted way. This results from the size dependence of the maximum induced transmembrane potential VMmax of a cell in an external electrical field E (radius R) according to VMmax=1.5*R*E and the practical size variation of biological objects. This is particularly problematic if different cell types are to be permeated and/or fused simultaneously, or only a few starting cells are available. Finally, the typical electroporation devices do not allow reliable individual cell manipulation or permeation.
Furthermore, it is generally known that biological objects can be manipulated with high frequency electrical fields on the basis of negative or positive dielectrophoresis. This is especially implemented in Microsystems, as it is, for example, described by G. Fuhr et al. in xe2x80x9cNaturwissenschaftenxe2x80x9d, vol. 81, 1994, p. 528 et seq. Thus, at a typical solvent conductivity of approximately 0.3 S/m, biological cells, under the effect of high frequency electrical fields over a large frequency range from approximately 1 MHz to over 120 MHz, show negative dielectrophoresis, i.e. the cells are moved with the electrodes having high frequency fields applied to them to regions of lower field strength. In culture media with conductivities over 1 S/m, animal cells show negative dielectrophoresis over all frequencies.
A device for cell fusion is known from JP 60-251876, in which the biological cells are positioned between planar electrodes under the effect of electrophoretic forces and fused by means of high-voltage treatment. The electrodes are on the channel walls of a microsystem. For fusing, the cells are affixed to the electrode surfaces. The microsystem has small enough dimensions that the cells positioned on opposing electrodes are in mutual contact. This fusion technology has several disadvantages. The positioning of the cells on the electrodes and their removal without leaving residue after fusing are difficult and time-consuming. A specific channel structure can always only be used for a specific cell size. The fusing is not reproducible, because multiple cells may collect between the electrodes.
A flow-through system for cell fusion and for nucleic acid transfer is known from JP 63-152971, in which electrode plates are affixed to two walls of a flow-through chamber. The electrode plates can, depending on the application, have direct current and alternating current voltages applied to them in order to subject cells which are washed through the flow-through chamber to an electrical treatment. The flow-through chamber of this system is manually detachable. It has a size which is significantly larger than the cells to be treated and therefore the same disadvantages as the typical electroporation devices mentioned above with closed chambers (without flow-through). A further disadvantage results in the flow-through system due to the unreproducible, undefined position of the objects to be treated. Correspondingly, no reproducible fusion results can be achieved either.
It is the object of the invention to provide an improved device for manipulation or treatment (particularly through permeation) of microscopic objects, whose field of application in regard to the selection of the ambient or culture media, the optimization of the poration parameter, and/or the handling of the smallest amounts of objects (down to individual objects) is expanded. The invention is particularly to allow performance of reproducible manipulation and/or treatment, e.g. corresponding to a defined protocol and possibly ensuring free observability. The object of the invention is also to provide an improved electroporation process using this type of device.
These objects are achieved by a device, an electroporation device, and/or a process with the features corresponding to patent claims 1, 8, 9 or 13, respectively. Advantageous embodiments of the invention arise from the dependent claims.
The basic idea of the invention consists of leaving the typical macroscopic electroporation arrangements toward microsystems, in which the object treatment is performed as the treatment of free suspended particles in ambient or culture media between miniaturized electrodes. According to a first important aspect of the invention, the electrodes are provided in a microsystem with a channel structure. In contrast to typical electroporation in closed cuvettes, the channel structure is set up as a flow-through system. The objects to be treated are thus guided by the streaming or flowing medium to the electrodes and permeated during flow-through or during a temporary fixed dielectrophoretic positioning of the objects in relation to the electrodes. During the electrical treatment of the objects, they are at a distance from the electrodes in the free suspension. The treatment occurs without contact with the electrodes and/or walls of the microsystem. According to a second important aspect of the invention, the objects are permeated at a sufficiently small distance from the electrodes that, even in highly conductive media, permeation can occur. Electrodes for exercising polarization forces on the basis of negative dielectrophoresis and electrodes for electric poration are provided in the microsystem.
According to a third important aspect of the invention, electrodes are provided which fulfill a double function. A device according to the invention has, e.g., an electrode system which is set up simultaneously to hold the objects in the medium or to guide the objects in a flowing medium and to apply electrical fields to the objects to realize electroporation. In contrast to typical electroporation devices, the electrodes according to the invention form a cage, closed in at least two spatial directions perpendicular to one another, in which the objects are manipulated and subjected to electroporation. The electrodes are set up to generate an inhomogenous electric field in the channel which has a minimum extending lengthwise in the flow direction. The objects to be treated are held in the field minimum with the electrodes, positioned continuously or with interruptions in the channel direction, which simultaneously are focusing and poration electrodes.
According to a preferred embodiment of the invention, the electrode system is set up in such a way that the poration of the objects occurs according to predetermined poration patterns. For this purpose, the electrodes (pulse electrodes) have field-forming devices such as electrode tips, which are positioned corresponding to the desired poration patterns, or shielding or covering elements, which allow exposure of the electrodes in regard to the medium with the desired poration patterns corresponding to the object(s).
A device according to the invention is preferably designed as a microsystem with channel structures which are equipped in at least one region with an electrode system according to the invention (electroporation region). These types of electroporation regions are advantageously combined in the microsystem with other regions for the treatment or manipulation of the objects, e.g. for collection or separation of specific object types (manipulation region). Microsystems according to the invention are preferably operated as flow-through systems.
The invention has the following advantages. Devices according to the invention allow object permeations in physiological solutions. The applicability of electroporation is thus expanded to media with higher conductivity (e.g. in the range from 0.01 to 10 S/m). For the first time, a reliable, contact-free, and protecting permeation of individual objects or groups of objects in a free solution is made possible. The miniaturizability of the system allows an increase of the electrode durability and a reduction of the electroporation pulse amplitudes (down to the volt to 100 V range), with the required high field strengths nonetheless being attainable. Treating the objects to be treated simultaneously at several positions according to predetermined defined poration patterns is made possible for the first time. A combination of the electroporation techniques, restricted to macroscopic applications until now, with procedural methods of microsystem technology is made possible. The object treatment according to the invention is performed without contact. Restrictions in regard to the adjustment of the microsystem to a specific object size are excluded. In addition, the object treatment is performed without residue. Contaminants on the electrodes are prevented.
Further advantages exist in the increased efficiency and yield of electroporation, the reduced heat production due to minimization of the electrode surface, the possibility of permeating objects of varying size, and the time-effective permeation of objects in flow-through systems at low voltages.
The invention is not restricted to biological cells, but can be appropriately implemented with all interesting synthetic formations with a membrane envelope, such as liposomes or vesicles, or with membrane or film materials.